Oligonucleotides, Use Thereof, Detecting Method and Kit for Diagnosing the Presence of H5 and N1 Genes of the Influenza a Virus

ABSTRACT

The present invention relates to a double pair of oligonucleotides for amplifying two target sequences located, respectively, in the H5 and N1 genes of the genome of the Influenza A virus, said oligonucleotides being of a length ranging between 10 and 50 nucleotides and comprising at least one fragment of 10 consecutive nucleotides derived from the following sequences: 
     
       
         
               
               
               
             
                   
                 SEQ ID No. 1: 
                 TGTATGTTGTGGAATGGCA, 
               
                   
                   
               
                   
                 SEQ ID No. 2: 
                 GCCGAATGATGCCATCAA, 
               
                   
                   
               
                   
                 SEQ ID No. 3: 
                 CGTGGATTGTCTCCGAAA, 
               
                   
                 and 
               
                   
                   
               
                   
                 SEQ ID No. 4: 
                 GGAATGCTCCTGTTATCCTGA 
               
           
              
              
              
              
              
              
              
              
             
          
         
       
         
         
           
             or the sequence complementary thereto. 
           
         
       
    
     The invention also relates to oligonucleotides for detecting amplicons, to the use of all these sequences, and to a method for detecting and a kit for diagnosing the presence of the H5 and N1 genes of the Influenza A virus. 
     The invention is particularly applicable in the field of diagnosis.

The present invention relates to a method for labeling nucleic acids inthe presence of at least one solid support.

The present invention relates to oligonucleotides for amplifying anddetecting two target sequences located, respectively, in the H5 and N1genes of the genome of the Influenza A virus.

The invention also relates to the use of these oligo-nucleotides, to amethod of detection and to a kit for diagnosing the presence of the H5and N1 genes of the Influenza A virus.

Among the conventional techniques routinely used for diagnosing flu,mention may be made of agglutination, inhibition of agglutination ordiffusion in agar gel. These methods are commonly used and make itpossible to characterize the flu virus A.

A possible alternative to these methods is viral culture in eggs at theembryonic stage or in MDCK cells, according to the protocol in themanual of the IOE [International Office for Epizootics]. However,several days are required in order to obtain the characterizationresults, this delay sometimes being incompatible with clinical needs oremergencies.

ELISA techniques for detecting antibodies or an antigen orimmunofluorescence tests have also been enormously developed, but these“immunological” methods of detection are often less sensitive and lessspecific than conventional viral culture.

The recent emergence of a highly pathogenic form of the bird flu virus,subtype H5N1, has therefore strongly relaunched the need for a rapid,specific and highly sensitive diagnostic test. For this reason, thevarious methods listed above have been replaced gradually with“molecular” techniques, such as the RT-PCR technique (reversetranscription associated with a polymerase chain reaction). RT-PCR,which is more sensitive, does not require the presence of “viable” virusin the samples, and thus makes it possible to type and subtype thevarious forms of the flu virus. The development of this real timetechnique (“real time RT-PCR”) has also greatly promoted its use fordiagnosing the type A virus.

For example, D. M. Whiley et al. describe, in a recent publication(Diagnostic Microbiology and Infectious Diseases (2005), in press), atest for detecting a broad spectrum of flu subtypes in clinical samples,based on an RT-PCR reaction involving a 5′-nuclease. Twooligonucleotides and a probe were chosen so as to be homologous to thegene encoding the M (matrix) protein of 23 subtypes of the A virus. Thistest thus makes it possible to detect the Influenza A virus in clinicalsamples, but does not, on the other hand, make it possible to preciselysubtype the form involved.

E. K. O, Ng et al. have recently disclosed (Emerging Infectious Diseases(2005) vol. 11 (8), p. 1303-1305) a test based on a multiplex RT-PCRreaction comprising two steps, using two sets of oligonucleotides and ofprobes that have been labeled in order to specifically target tworegions of the HA gene of H5N1. This has thus made it possible todevelop a rapid and sensitive test for directly detecting the H5 subtypein human samples. This test has been validated on clinical samplesoriginating from patients infected in Hong Kong and in Vietnam with theH5N1 subtype of the virus, but does not make it possible to diagnosedirectly which virus subtype is involved in the infection.

S. Payungporn et al. describe (Viral Immunology (2004) vol. 17 (4), p.588-593 and Journal of Virological Methods (2005), in press) a methodfor simultaneously detecting the M, H5 and N1 sequences of the H5N1subtype, based on a real time multiplex RT-PCR test and in a singlestep. Oligonucleotides corresponding to the M, H5 and N1 sequences andalso various labeled TaqMan probes were selected and used in the test inorder to simultaneously detect three fluorescent signals. The H5 and N1oligonucleotides were chosen from invariant regions covering more than50 known sequences specific for the H5N1 virus. However, it should benoted that this RT-PCR method, which is nonisothermal, is sometimesliable to contamination.

Another molecular method that can be used for detecting the presence ofan infectious agent is the NASBA technique (Nucleic Acid Sequence-BasedAmplification). For example, R. A. Collins et al. describe (Journal ofVirological Methods (2002) vol. 103, p. 213-225 and Avian Diseases(2003) vol. 47 (3), p. 1069-1074) the detection of the H5 subtype ofbird flu (detection of the highly pathogenic and weakly pathogenicsubtype) using the NASBA technique coupled to an ECL(electro-chemiluminescence) detection system. The NASBA technique is amethod of isothermal amplification of nucleic acids involving severalenzymatic activities, which allows rapid detection of the H5 virus. Theamplification of nucleic acids via this process is suitable for RNAgenomes, such as the genome of the flu virus, by virtue of theintroduction of the reverse transcription step directly into theamplification reaction. Nevertheless, even though the method describedin this publication makes it possible to detect and distinguish theweakly pathogenic strains from the highly pathogenic strains, it doesnot make it possible to specifically identify the H5N1 form of thevirus.

A test for identifying the Influenza A virus in its H5N1 form using atranscriptional amplification technique, which is particularly suitablefor amplifying and detecting RNA viruses, is therefore still awaited. Inaddition, a multiplex test, i.e. a test which makes it possible tosimultaneously amplify and detect the two H5 and N1 genes, theamplification technique being a transcriptional amplification techniquesuch as NASBA, is particularly advantageous.

The present invention therefore relates to a double pair ofoligonucleotides for amplifying two target sequences located,respectively, in the H5 gene and in the N1 gene of the genome of theInfluenza A virus, the pair of oligonucleotides for amplifying the H5gene consisting of:

-   -   a first oligonucleotide of a length ranging between 10 and 50        nucleotides and comprising at least one fragment of 10        consecutive nucleotides derived from:    -   SEQ ID No. 1: TGTATGTTGTGGAATGGCA, or the sequence complementary        thereto, and    -   a second oligonucleotide of a length ranging between 10 and 50        nucleotides and comprising at least one fragment of 10        consecutive nucleotides derived from:    -   SEQ ID No. 2: GCCGAATGATGCCATCAA, or the sequence complementary        thereto,        while the pair of oligonucleotides for amplifying the N1 gene        consists of:    -   a first oligonucleotide of a length ranging between 10 and 50        nucleotides and comprising at least one fragment of 10        consecutive nucleotides derived from:    -   SEQ ID No. 3: CGTGGATTGTCTCCGAAA, or the sequence complementary        thereto, and    -   a second oligonucleotide of a length ranging between 10 and 50        nucleotides and comprising at least one fragment of 10        consecutive nucleotides derived from:    -   SEQ ID No. 4: GGAATGCTCCTGTTATCCTGA, or the sequence        complementary thereto.

The invention may also relate to a pair of oligonucleotides foramplifying a target sequence located in the H5 gene of the genome of theInfluenza A virus, the pair of oligonucleotides consisting of:

-   -   a first oligonucleotide of a length ranging between 10 and 50        nucleotides and comprising at least one fragment of 10        consecutive nucleotides derived from:    -   SEQ ID No. 1: TGTATGTTGTGGAATGGCA, or the sequence complementary        thereto, and    -   a second oligonucleotide of a length ranging between 10 and 50        nucleotides and comprising at least one fragment of 10        consecutive nucleotides derived from:    -   SEQ ID No. 2: GCCGAATGATGCCATCAA, or the sequence complementary        thereto.

Similarly, the invention can also cover a pair of oligonucleotides foramplifying a target sequence located in the N1 gene of the genome of theInfluenza A virus, the pair of oligonucleotides consisting of:

-   -   a first oligonucleotide of a length ranging between 10 and 50        nucleotides and comprising at least one fragment of 10        consecutive nucleotides derived from:    -   SEQ ID No. 3: CGTGGATTGTCTCCGAAA, or the sequence complementary        thereto, and    -   a second oligonucleotide of a length ranging between 10 and 50        nucleotides and comprising at least one fragment of 10        consecutive nucleotides derived from:    -   SEQ ID No. 4: GGAATGCTCCTGTTATCCTGA, or the sequence        complementary thereto.

In the three situations above, in the pair(s) of oligonucleotides, thefirst oligonucleotide additionally comprises a promoter sequence whichcan be recognized by a DNA-dependent RNA polymerase enzyme.

More specifically, the promoter sequence which can be recognized by aDNA-dependent RNA polymerase enzyme is a T7 polymerase.

In relation to the above two cases, when this oligonucleotide, to whichthe promoter sequence is added, makes it possible to amplify the targetsequence located in the H5 gene, it essentially consists of thefollowing sequence:

SEQ ID No. 5: aattctaatacgactcactataggggTGTATGTTGTGGAATGGCA, or thesequence complementary thereto. The part of the sequence in lower caseletters corresponds to the T7 promoter sequence.

In the same manner as above, when this oligonucleotide makes it possibleto amplify the target sequence located in the N1 gene it essentiallyconsists of the following sequence:

SEQ ID No. 6: aattctaatacgactcactataggggCGTGGATTGTCTCCGAAA, or thesequence complementary thereto.

In all situations, each oligonucleotide may be of a length ranging frombetween 12 and 30 nucleotides and comprising at least one fragment of 16consecutive nucleotides, and preferably of a length ranging between 15and 26 nucleotides and comprising at least one fragment of 18consecutive nucleotides.

The invention also relates to a pair of oligo-nucleotides for use as aprobe for detecting two target sequences located, respectively, in theH5 and N1 genes of the genome of the Influenza A virus, the probe fordetecting the H5 gene consisting of:

SEQ ID No. 7: ACACCAAGTGTCAAACTCCAAT, or the sequence complementarythereto,while the probe for detecting the N1 gene consists of:SEQ ID No. 8: GCGAAATCACATGTGTGTGCAGGGA, or the sequence complementarythereto,each sequence comprising at least one labeling means.

The invention may also relate to an oligonucleotide for use as a probefor detecting an amplified nucleic acid sequence resulting from theamplification of a target sequence located in the H5 gene of the genomeof the Influenza A virus, said amplification being carried out by meansof a pair of oligonucleotides as described above, the detection probebeing of a length ranging between 10 and 50 nucleotides and comprisingat least one fragment of 10 consecutive nucleotides derived from:

SEQ ID No. 7: ACACCAAGTGTCAAACTCCAAT, or the sequence complementarythereto, the sequence comprising at least one labeling means.

When it is desired to detect the N1 gene of the genome of the InfluenzaA virus, the invention recommends an oligonucleotide for use as a probefor detecting an amplified nucleic acid sequence resulting from theamplification of a target sequence located in this N1 gene, saidamplification being carried out by means of a pair of oligonucleotidesas described above, the detection probe being of a length rangingbetween 10 and 50 nucleotides and comprising at least one fragment of 10consecutive nucleotides derived from:

SEQ ID No. 8: GCGAAATCACATGTGTGTGCAGGGA, or the sequence complementarythereto, the sequence comprising at least one labeling means.

In an advantageous embodiment of the invention, the detection probeconsists of a “molecular beacon” subsequently known as molecular probe.The molecular probes are detection probes in the form of single-strandedoligonucleotides, which have a stem loop structure well-known to thoseskilled in the art. The loop contains a probe sequence complementary tothe target sequence (amplicon in general), and the stem is formed by thehybridization of two sequences forming arms, which are each located ateach end of the probe. A fluorophore is covalently bonded to the end ofone of the two arms and a quencher (fluorescence absorber) is covalentlybonded to the end of the other arm. The molecular probes do notfluoresce when they are free in solution. However, in the presence ofcomplementary amplicons, when they hybridize to these targets, theyundergo a conformational change which allows them to fluoresce. In theabsence of targets, the stem keeps the fluorophore in close proximitywith the quencher, thereby leading to the transfer of the fluorescencefrom the fluorophore to the quencher. Said quencher is a nonfluorescentchromophore which dissipates the energy received from the fluorophore asheat. When the probe encounters a molecular target, a probe-targethybrid is formed, which hybrid is longer and more stable than the hybridcreated by the two arms of the stem. The rigidity and the length of theprobe-hybrid hybrid prevent the simultaneous existence of the stemhybrid. Consequently, the molecular probe undergoes spontaneousconformational reorganization which forces the stem hybrid to dissociateand the fluorophore and the quencher to move away from one another,thereby restoring the fluorescence.

More specifically, the detection probe consists of a molecular probepreferably composed of:

SEQ ID No. 9: [6-FAM]-cgatcgACACCAAGTGTCAAACTCCAATcgatcg-[DabSyl], fordetecting the H5 gene,SEQ ID No. 10: [6-FAM]-cgatcgGCGAAATCACATGTGTGTGCAGGGAcgatcg-[DabSyl],for detecting the N1 gene.

When the test that it is desired to carry out is a multiplex test wherethe detection of the H5 and N1 genes is simultaneous and carried out ina single container, it is advisable to use two different labels. Amongthe possible fluorescent labels, mention may be made, in a nonlimitingmanner, of:

-   -   fluorescein (FAM)    -   tetrachloro-6-carboxyfluorescein (TET)    -   tetramethylrhodamine (TMR)    -   5-carboxyrhodamine 6G (RHD)    -   carboxyrhodamine (ROX), and    -   cyanin 5 (CY5).

Each of the two sequences SEQ ID Nos. 9 and 10 above will therefore beprovided with one of these labels, the two labels used being differentfrom one another in order to allow differentiation between the detectedsignals.

The invention also proposes the use of one or two pairs ofoligonucleotides, as described above, in a reaction for theamplification of nucleic acids or as a probe for the detection of thegenome of the Influenza A virus suspected of being present in abiological sample.

The invention also relates to a method for detecting nucleic acids ofthe Influenza A virus that may be present in a sample, in which thesample is subjected to a reaction for the amplification of nucleic acidsusing a pair of oligonucleotides, as described above, in the presence ofthe amplification reagents required for such an amplification, and thepresence of amplicons of interest is detected.

This method of detection can be based on an RT-PCR amplificationreaction.

Alternatively, this method of detection can be based on atranscriptional amplification technique. Preferably, this technique isthe NASBA technique.

The invention also relates to a method for amplifying two H5 and N1genes of the Influenza A virus that may be present in a sample,comprising the following steps:

-   -   incubating the sample in an amplification buffer in the        presence:        -   of two amplification primers, each having a length ranging            between 10 and 50 nucleotides, one additionally comprising a            promoter sequence, the other of opposite polarity to the            primer associated with the promoter sequence, in order to            hybridize respectively upstream and downstream of a region            of interest located in the H5 gene of the Influenza A virus,        -   of two amplification primers, each having a length ranging            between 10 and 50 nucleotides, one additionally comprising a            promoter sequence, the other of opposite polarity to the            primer associated with the promoter sequence, in order to            hybridize respectively upstream and downstream of a region            of interest located in the N1 gene of the Influenza A virus,    -   adding the following reagents to the sample:        -   an enzyme having an RNA-dependant DNA polymerase activity,        -   an enzyme having a DNA-dependant DNA polymerase activity,        -   an enzyme having an RNase H activity,        -   an enzyme having a DNA-dependant RNA polymerase activity,            and    -   maintaining the reaction mix thus created under suitable        conditions and for a period of time sufficient for an        amplification to take place.

There are four enzymes listed above, but it is entirely possible to makeuse of one enzyme having two or even three of the abovementionedactivities; in this case the use of three or even of two enzymes remainspossible and covered by the invention. Furthermore, other elementsrequired to establish an amplification are necessary, such asnucleotides. Such elements are well-known to those skilled in the art.

Finally, the invention proposes a kit for detecting the H5 and N1 genesof the Influenza A virus that may be present in a sample, containing:

-   -   two pairs of amplification oligonucleotides or primers as        defined above for carrying out the amplification of the two        regions of interest H5 and N1,    -   two oligonucleotides that are labeled or that can be labeled, as        described above, and that have a nucleic acid sequence        substantially complementary with at least one part of the        amplified H5 or N1 nucleic acid sequence,    -   reagents required for carrying out an amplification reaction.

The term “substantially complementary” is intended to mean that ahybridization is carried out between an oligonucleotide that is labeledor that can be labeled, otherwise referred to as detection probe, and atleast one part of the amplified nucleic acid sequence or amplicon, thishybridization being sufficiently specific and selective to allow thedetection of the amplicon of interest.

Furthermore, the reagents required for carrying out an amplificationreaction are reagents for a NASBA amplification.

The term “detectable label” is intended to mean at least one labelcapable of directly generating a detectable signal. For example, thepresence of biotin is considered to be direct labeling, since it isdetectable even though it is possible to subsequently associate it withlabeled streptavidin. A non-limiting list of these labels follows:

-   -   enzymes which produce a signal that can be detected, for        example, by colorimetry, fluorescence or luminescence, such as        horseradish peroxydase, alkaline phosphatase, β-galactosidase or        glucose-6-phosphate dehydrogenase,    -   chromophores such as fluorescent, luminescent or dye compounds,    -   electron dense groups that can be detected by electron        microscopy or by virtue of their electrical properties such as        conductivity, amperometry, voltametry or impedance,    -   detectable groups, for example, the molecules of which are of        sufficient sizes to induce detectable modifications of their        physical and/or chemical characteristics; this detection can be        carried out by optical methods such as diffraction, surface        plasmon resonance, surface variation, contact angle variation,        or physical methods such as atomic force spectroscopy, or tunnel        effect,    -   radioactive molecules such as ³²P, ³⁵S or ¹²⁵I.

Preferably, the label is not a radioactive label, so as to avoid thesafety problems associated with these labels.

In a specific embodiment of the present invention, the label isdetectable electrochemically, and in particular the label is aderivative of an iron complex, such as a ferrocene.

The term “nucleic acid” means a series of at least twodeoxyribonucleotides or ribonucleotides, optionally comprising at leastone modified nucleotide, for example, at least one nucleotide comprisinga modified base, such as inosine, methyl-5-deoxycytidine,dimethylamino-5-deoxyuridine, deoxyuridine, diamino-2,6-purine orbromo-5-deoxyuridine, or any other modified base that allowshybridization. This polynucleotide can also be modified at the level ofthe internucleotide bond, for instance phosphorothioates, H-phosphonatesor alkyl phosphonates, or at the level of the backbone, for instance,alpha-oligonucleotides (FR 2 607 507) or PNA (M. Egholm et al., J. Am.Chem. Soc., 114, 1895-1897, 1992) or 2′-O-alkyl riboses and LNAs (B W.Sun et al., Biochemistry, 4160-4169, 43, 2004). The nucleic acid may benatural or synthetic, an oligonucleotide, a polynucleotide, a nucleicacid fragment, a ribosomal RNA, a messenger RNA, a transfer RNA, or anucleic acid obtained by means of an enzymatic amplification techniquesuch as:

-   -   PCR (Polymerase Chain Reaction), described in U.S. Pat. No.        4,683,195, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,800,159        and its derivative RT-PCR (Reverse Transcription PCR), in        particular in a one-step format, as described in patent        EP-B-0.569.272,    -   LCR (Ligase Chain Reaction), disclosed, for example in patent        application EP-A-0.201.184,    -   RCR (Repair Chain Reaction), described in patent application        WO-A-90/01069,    -   3SR (Self Sustained Sequence Replication) with patent        application WO-A-90/06995,    -   NASBA (Nucleic Acid Sequence-Based Amplification) with patent        application WO-A-91/02818,    -   TMA (Transcription Mediated Amplification) with U.S. Pat. No.        5,399,491, and    -   RCA (Rolling Circle Amplification) (U.S. Pat. No. 6,576,448).

The term amplicons is then used to denote the nucleic acids generated byan enzymatic amplification technique.

Each of these modifications can be taken in combination.

The amplification and detection steps disclosed above can be preceded bya purification step. The term “purification step” is intended to mean inparticular the separation between the nucleic acids of themicroorganisms and the cellular constituents released in the lysis stepwhich precedes the nucleic acid purification. These lysis steps arewell-known; by way of indicative example, use may be made of the lysismethods as described in patent applications:

-   -   WO-A-00/60049 on lysis by sonication,    -   WO-A-00/05338 on mixed magnetic and mechanical lysis,    -   WO-A-99/53304 on electrical lysis, and    -   WO-A-99/15621 on mechanical lysis.

Those skilled in the art may use other well-known methods of lysis, suchas heat shock or osmotic shock or treatments with chaotropic agents,such as guanidium salts (U.S. Pat. No. 5,234,809).

This step generally makes it possible to concentrate the nucleic acids.By way of example, it is possible to use solid supports, such asmagnetic particles (in this respect, see U.S. Pat. No. 4,672,040 andU.S. Pat. No. 5,750,338), and thus to purify the nucleic acids, whichare attached to these magnetic particles, by means of a washing step.This nucleic acid purification step is particularly advantageous if itis desired to subsequently amplify said nucleic acids. A particularlyadvantageous embodiment of these magnetic particles is described inpatent applications WO-A-97/45202 and WO-A-99/35500.

The term “solid support” as used here includes all materials to which anucleic acid can be attached. Synthetic materials or natural materials,which have been optionally chemically modified, can be used as solidsupport, in particular polysaccharides, such as cellulose-basedmaterials, for example, paper, cellulose derivatives such as celluloseacetate and nitrocellulose or dextran; polymers, copolymers, inparticular based on styrene-type monomers, natural fibers such ascotton, and synthetic fibers such as nylon; mineral materials such assilica, quartz, glasses, ceramics; latices; magnetic particles; metalderivatives, gels, etc. The solid support may be in the form of amicrotitration plate, of a membrane, of a particle or of a substantiallyflat glass or silicon plate, or derivatives.

It is possible to carry out the entire protocol (from the sample takento the amplicons ready to be hybridized) in one and the same tube,processed manually or in an automated machine.

The attached examples represent specific embodiments and cannot beconsidered to limit the scope of the present invention.

Several controls were carried out in these examples. First of all, anegative control with water; in this case, no signal for either H5 orfor N1 is detected. Secondly, a specificity control with Influenza H3N2RNA; here again, no signal was detected for either H5 or for N1.

EXAMPLE 1 Experiment to Evaluate the H5N1 Primers on an H5N1 RNAOriginating from a Clinical Sample from Asia

The sequences of the pairs of oligonucleotides (N1_P1 and N2_P2, usedfor amplifying and detecting the N1 sequence, and H5_P1 and H5_P2, usedfor amplifying and detecting the H5 sequence) and of the detectionprobes present in the form of molecular probes (molecular probe N1 andmolecular probe H5) are indicated below:

N1 P2 5′-GGAATGCTCCTGTTATCCTGA-3′ N1 P15′-AATTCTAATACGACTCACTATAGGGGCGTGGATTGTCTC CGAAA3′ N1 probe5′-CGATCGGCGAAATCACATGTGTGTGCAGGGACGATCG- 3′ H5 P25′-GCCGAATGATGCCATCAA-3′ H5 P1 5′-AATTCTAATACGACTCACTATAGGGGTGTATGTTGTGGAATGGCA-3′ H5 probe 5′-CGATCGACACCAAGTGTCAAACTCCAATCGATCG-3′

The sequence indicated in bold corresponds to the T7 promoter sequence,recognized by the T7 RNA polymerase, and is found in the P1oligonucleotides for carrying out the NASBA technique.

The sample is processed using the miniMAG system, as described in theoperating protocol. The kits used are:

-   -   NucliSens Lysis Buffer, bioMérieux B. V. (Boxtel, Holland),        Batch No. 200295, and    -   NucliSens Magnetic Extraction Reagents, bioMérieux B. V., Batch        No. #200297.

Operating Protocol for the Detection Test:

According to the instructions of the NucliSens EasyQ Basic Kit(bioMérieux B. V., Boxtel, Holland, Batch No. #285006) two reactionmixes, one serving to amplify and detect the H5 sequence (“mix H5”) andthe other serving to amplify and detect the N1 sequence (“mix N1”) wereprepared. Briefly, 11 μl of water, 13 μl of KCl at 1.2 M, 4 μl of eacholigonucleotide (H5_P1 and H5_P2 or N1_P1 and N1_P2, stock solution at10 μM) and 0.8 μl of the appropriate detection probe (the molecularprobe H5 or the molecular probe N1, stock solution at 2 μM) were addedto 64 μl of diluent. A volume of 10 μl of each mix was then added to 5μl of the RNA target. In parallel, a solution of enzymes was prepared,and 5 μl of this solution were added to the reaction mix in the tube fora final volume of 20 μl. The samples were then placed under the reactionconditions recommended by the NucliSens EasyQ Basic Kit in order toallow the amplification and detection of the sequences of interest bythe isothermal NASBA technique (Kievits, T et al. J. Virol. Methods(1991) vol. 35(3), p. 273-286).

In order to validate this detection test, the RNA targets used were thefollowing:

-   -   H5N1: Influenza A subtype H5N1 Vietnam 1194/2004    -   Influenza A: subtype H3N2,    -   H5 control RNA: subtype H5N3,    -   Influenza B.

The appropriate (positive and negative) controls were included in thetest.

After amplification and detection on the NucliSens EasyQ system, thefollowing results were obtained:

NASBA H5 NASBA N1 Virus Results Results Negative control NegativeNegative Influenza A H3N2 Negative Negative Negative Negative NegativeFlu A H5N1 Vietnam 1194/2004 Positive Positive Negative control NegativeNegative Flu A H5N1 Vietnam 1194/2004 Positive Positive Flu A H5N3 Duckcontrol virus Positive Negative

These results show that the H5N1 detection probes and oligonucleotidesare specific and indeed detect their respective targets H5 and N1.

EXAMPLE 2 Experiment to Evaluate the Detection Probes and Primers forH5N1 in a Multiplex Test

In the case of the multiplex, the molecular probe N1 used is labeledwith CY5, whereas the molecular probe H5 remains labeled with FAM.

For information, in order to demonstrate the functionality of oursequences in a multiplex test, the target used is a synthetictranscript, which was constructed from the recombinant H5N1 RNA. It isan RNA, which comprises only the H5 and N1 regions. It is used asreference RNA for evaluating the performance levels of our amplificationprimers and detection probes for H5 and N1 (since it is a synthetictranscript, it is available in large amount, which means that therecombinant H5N1 RNA, which is very precious, does not have to be used).

According to the instructions of the NucliSens EasyQ Basic Kit(bioMérieux B. V., Boxtel, Holland, Batch No. #285006), a singlereaction mix for simultaneously detecting the H5 gene and the N1 gene isprepared. Briefly, 11 μl of water, 13 μl of KCl at 1.2 M, 8 μl of asolution of oligonucleotides and of molecular probes (containing theoligonucleotides H5_P1 and H5_P2 for H5 at 5 μM, the oligonucleotidesN1_P1 and N1_P2, for N1 at 20 μM, the molecular probe H5-FAM at 2 μM andthe molecular probe N1-CY5 at 2 μM) were added to 64 μl of diluent. Avolume of 10 μl of the mix was then added to 5 μl of the RNA target(H5N1 transcript at 1000 copies/NASBA).

The difference in concentration between the H5 primers and the N1primers is approximately inversely proportional to the difference insensitivity. The concentration of H5 primers is therefore four timeslower than that of N1 primers.

In parallel, a solution of enzymes was prepared, and 5 μl of thissolution were added to the reaction mix in the tube, for a final volumeof 20 μl. The samples were then placed under the reaction conditionsrecommended by the NucliSens EasyQ Basic Kit in order to allow theamplification and detection of the sequences of interest by theisothermal NASBA technique (Kievits, T et al. J. Virol. Methods (1991)vol. 35(3), p. 273-286). The results can be seen in FIG. 1. Theseresults show that the simultaneous detection of H5 and N1 in a singletube functions very well on a synthetic transcript.

1. A double pair of oligonucleotides for amplifying two target sequenceslocated, respectively, in the H5 gene and in the N1 gene of the genomeof the Influenza A virus, the pair of oligonucleotides for amplifyingthe H5 gene consisting of: a first oligonucleotide of the length rangingbetween 10 and 50 nucleotides and comprising at least one fragment of 10consecutive nucleotides derived from: SEQ ID No. 1: TGTATGTTGTGGAATGGCA,or the sequence complementary thereto, and a second oligonucleotide of alength ranging between 10 and 50 nucleotides and comprising at least onefragment of 10 consecutive nucleotides derived from: SEQ ID No. 2:GCCGAATGATGCCATCAA, or the sequence complementary thereto, while thepair of oligonucleotide for amplifying the N1 gene consists of: a firstoligonucleotide of a length ranging between 10 and 50 nucleotides andcomprising at least one fragment of 10 consecutive nucleotides derivedfrom: SEQ ID No. 3: CGTGGATTGTCTCCGAAA, or the sequence complementarythereto, and a second oligonucleotide of a length ranging between 10 and50 nucleotides and comprising at least one fragment of 10 consecutivenucleotides derived from: SEQ ID No. 4: GGAATGCTCCTGTTATCCTGA, or thesequence complementary thereto.
 2. A pair of oligonucleotides foramplifying a target sequence located in the H5 gene of the genome of theInfluenza A virus, the pair of oligonucleotides consisting of: a firstoligonucleotide of a length ranging between 10 and 50 nucleotides andcomprising at least one fragment of 10 consecutive nucleotides derivedfrom: SEQ ID No. 1: TGTATGTTGTGGAATGGCA, or the sequence complementarythereto, and a second oligonucleotide of a length ranging between 10 and50 nucleotides comprising at least one fragment of 10 consecutivenucleotides derived from: SEQ ID No. 2: GCCGAATGATGCCATCAA, or thesequence complementary thereto.
 3. A pair of oligonucleotides foramplifying a target sequence located in the N1 gene of the genome of theInfluenza A virus, the pair of oligonucleotides consisting of: a firstoligonucleotide of a length ranging between 10 and 50 nucleotides andcomprising at least one fragment of 10 consecutive nucleotides derivedfrom: SEQ ID No. 3: CGTGGATTGTCTCCGAAA, or the sequence complementarythereto, and a second oligonucleotide of a length ranging between 10 and50 nucleotides and comprising at least one fragment of 10 consecutivenucleotides derived from: SEQ ID No. 4: GGAATGCTCCTGTTATCCTGA, or thesequence complementary thereto.
 4. The pair(s) of oligonucleotides asclaimed in claim 1, wherein the first oligonucleotide additionallycomprises a promoter sequence which can be recognized by a DNA-dependentRNA polymerase enzyme.
 5. The pair(s) of oligonucleotides as claimed inclaim 4, wherein the promoter sequence which can be recognized by aDNA-dependent RNA polymerase enzyme is a T7 polymerase.
 6. The pair ofoligonucleotides as claimed in claim 4, in which the firstoligonucleotide, when this oligonucleotide makes it possible to amplifythe target sequence located in the H5 gene, is wherein it consistsessentially of the following sequence: SEQ ID No. 5:AATTCTAATACGACTCACTATAGGGGTGTATGTTGTGGAATGGCA, or the sequencecomplementary thereto.
 7. The pair of oligonucleotides as claimed inclaim 4, in which the first oligonucleotide, when this oligonucleotidemakes it possible to amplify the target sequence located in the N1 gene,consists essentially of the following sequence: SEQ ID No. 6:AATTCTAATACGACTCACTATAGGGGCGTGGATTGTCTCCGAAA, or the sequencecomplementary thereto.
 8. The pair of oligonucleotides as claimed inclaim 1, wherein each oligonucleotide is of a length ranging between 12and 30 nucleotides and comprising at least one fragment of 16consecutive nucleotides, and preferably of a length ranging between 15and 26 nucleotides and comprising at least one fragment of 18nucleotides.
 9. A pair of oligonucleotides for use a probe for detectingtwo target sequences located, respectively in the H5 and N 1 genes ofthe genome of the Influenza A virus, the probe for detecting the H5 geneconsisting of SEQ ID No. 7: ACACCAAGTGTCAAACTCCAAT, or the sequencecomplementary thereto, while the probe for detecting the N1 geneconsists of: SEQ ID No. 8: GCGAAATCACATGTGTGTGCAGGGA, or the sequencecomplementary thereto, each sequence comprising at least one labelingmeans.
 10. An oligonucleotide for use as a probe for detecting anamplified nucleic acid sequence resulting from the amplification of atarget sequence located in the H5 gene of the genome of the Influenza Avirus, said amplification being carried out by means of a pair ofoligonucleotides as claimed in claim 2, the detection probe being of alength ranging between 10 and 50 nucleotides and comprising at least onefragment of 10 consecutive nucleotides derived from: SEQ ID No. 7:ACACCAAGTGTCAAACTCCAAT, or the sequence complementary thereto, thesequence comprising at least one labeling means.
 11. An oligonucleotidefor use as a probe for detecting an amplified nucleic acid sequenceresulting from the amplification of a target sequence located in the N1gene of the genome of the Influenza A virus, said amplification beingcarried out by means of a pair of oligonucleotides as claimed in claim3, the detection probe being of a length ranging between 10 and 50nucleotides and comprising at least one fragment of 10 consecutivenucleotides derived from: SEQ ID No. 8: GCGAAATCACATGTGTGTGCAGGGA, orthe sequence complementary thereto, the sequence comprising at least onelabeling means.
 12. The detection probe as defined in claim 9, whereinit is composed of a molecular probe.
 13. The detection probe as claimsin claim 9, wherein it is composed of a molecular probe, preferablycomposed of: SEQ ID 9:[6-FAM]-cgatcgaCACCAAGTGTCAAACTCCAAtcgatcg-[DabSyl] for detecting the H5gene, SEQ ID 10: [6-FAM]-cgatcgaCACCAAGTGTCAAACTCCAAtcgatcg-[DabSyl] fordetecting the N1 gene.
 14. The use of one or two pairs ofoligonucleotides, as claimed in claim 8, in a reaction for theamplification of nucleic acids or as a probe for the detection of thegenome of the Influenza A virus suspected of being present in abiological sample.
 15. A method for detecting nucleic acids of theInfluenza A virus that may be present in a sample, in which the sampleis subjected to a reaction for the amplification of nucleic acids usinga pair of oligonucleotides, as claimed in claim 1, in the presence ofthe amplification reagents required for such an amplification, and thepresence of amplicons of interest is detected.
 16. The method as claimedin claim 15, wherein the amplification reaction used is an RT-PCR. 17.The method as claimed in claim 15, wherein the amplification reactionused is a transcriptional amplification technique.
 18. The method asclaimed in claim 17, wherein the amplification reaction used is theNASBA technique.
 19. A method for amplifying two H5 and N1 genes of theInfluenza A virus that may be present in a sample, comprising thefollowing steps: incubating the sample in an amplification buffer in thepresence: of two amplification primers, each having a length rangingbetween 10 and 50 nucleotides, one additionally comprising a promotersequence, the other of opposite polarity to the primer associated withthe promoter sequence, in order to hybridize respectively upstream anddownstream of a region of interest located in the H5 gene of theInfluenza A virus, of two amplification primers, each having a lengthranging between 10 and 50 nucleotides, one additionally comprising apromoter sequence, the other of opposite polarity to the primerassociated with the promoter sequence, in order to hybridizerespectively upstream and downstream of a region of interest located inthe N1 gene of the Influenza A virus, adding the following reagents tothe sample: an enzyme having an RNA-dependant DNA polymerase activity,an enzyme having a DNA-dependant DNA polymerase activity, an enzymehaving an Rnase H activity, an enzyme having a DNA-dependent RNApolymerase activity, and maintaining the reaction mix thus created undersuitable conditions and for a period of time sufficient for anamplification to take place.
 20. A kit for detecting the H5 and N1 genesof the Influenza A virus that may be present in a sample, containing:two pairs of oligonucleotides as claimed in claim 1, twooligonucleotides that are labeled or that can be labeled, for use aprobe for detecting two target sequences located, respectively in the H5and N1 genes of the genome of the Influenza A virus, the probe fordetecting the H5 gene consisting of SEQ ID No. 7:ACACCAAGTGTCAAACTCCAAT, or the sequence complementary thereto, while theprobe for detecting the N1 gene consists of: SEQ ID No. 8:GCGAAATCACATGTGTGTGCAGGGA, or the sequence complementary thereto, eachsequence comprising at least one labeling means and that have a nucleicacid sequence substantially complementary with at least one part of theamplified nucleic acid sequence, reagents required for carrying out anamplification reaction.
 21. The kit as claimed in claim 20, in which thereagents required for carrying out an amplification reaction arereagents for a NASBA amplification.